一种结肠类器官的提取分离方法

A Method for Extraction and Separation of Colon Organoids

类器官是一种来源于干细胞,在特定诱导条件下分化为与研究器官细胞组成类似的3D培养物。与传统细胞培养物相比,类器官更加接近研究器官的三维结构和特征,因此基于类器官进行研究更能体现所研究器官体内的真实反应。这一研究手段广泛应用于肿瘤药物筛选,具有广阔前景。文章目的是建立一种稳定的结肠类器官提取分离方法,使得到的类器官有良好的增殖活性;建立结肠类器官增殖活性评价方法,能够准确评价结肠类器官的增殖活性。采用方法为取大约5 cm C57BL/6小鼠的结肠,用胶原酶分离结肠隐窝,通过梯度离心纯化隐窝,最后将其用基质胶包埋接种在培养皿中,加入肠道类器官培养基培养6 d,最后使用光学显微镜拍照记录并量化类器官的出芽情况,通过qPCR检测干细胞标志物Lgr5的表达。结果显示提取得到的类器官较为纯净,通过光学显微镜可以记录类器官的出芽情况,计算平均每个类器官含有的隐窝数来量化类器官增殖活性,qPCR检测发现干细胞标志物Lgr5的表达与平均每个类器官含有的隐窝数变化一致,因此可以通过出芽情况和基因表达情况来表征类器官的增殖活性。文章建立的结肠类器官提取和分析方法可以用于研究在不同条件影响下结肠类器官的增殖变化,还可以研究药物对结肠类器官的影响机制,可以作为结肠体外研究的有力工具。

Organoid is a kind of 3D culture derived from stem cells and differentiated into cells with similar composition to the study organ under specific induction conditions.Compared with the traditional cell culture,organoid is closer to the 3D structure and characteristics of the research organ,so the research based on organoid can better reflect the real reaction in vivo of the studied organ.This research method is widely used in tumor drug screening and has broad prospects.To establish a stable method for extraction and separation of colon organoids,so that the obtained organoids have good proliferative activity.To establish a method for evaluating the proliferative activity of colon organs,which can accurately evaluate the proliferative activity of colonic organs.Take about 5 cm colon tissue from C57BL/6 mice,isolate the colonic crypt with collagenase,purify the crypt through gradient centrifuga⁃tion,and finally embed it in a culture dish with matrigel,add it to the intestinal organoids culture medium for 6 d.Finally,take photos with optical microscope to record and quantify the budding of organoids,and detect the expression of stem cell marker gene Lgr5 by qPCR.The extracted organoids were relatively clean.The budding of organoids could be recorded through the optical microscope.Crypt per organoid was calculated to quantify the proliferative activity of the organoids.The qPCR detection result found that the expression of the stem cell marker gene Lgr5 was consistent with Crypt per organoid.Therefore,the proliferative activity of organoid can be characterized by budding and gene expression.The method of extraction and analysis of colon organs established in this paper can be used to study the proliferation changes of colonic organs under different conditions,and also can study the mechanism of drug effects on colonic organs,which can be used as a powerful tool for colon in vitro research.